AT397513B - AMPEROMETRIC ENZYME ELECTRODE - Google Patents

Description

AT 397 513 B

The invention relates to an amperometric enzyme electrode for measuring the concentration of an enzyme substrate in a sample with an enzyme immobilized or adsorbed on or in an electrically conductive, porous electrode material, the electrode material comprising a redox-inactive conductor with a conductive pigment and a non-conductive binder and has a finely divided catalytic substance therein.

The determination of substrates by amperometric biosensors or enzyme electrodes based on hydrogen peroxide measurement with immobilized oxidoreductases is the subject of a large number of patents and publications and plays a not inconsiderable role in biochemical sensors. When measuring substrates such. B. glucose in body fluids (blood, serum, interstitial fluid and ham) raises the problem of interference. For example, many redox-active metabolites and drugs adulterate. B. p-acetamol, ascorbate, uric acid and the like. a. the substrate determination according to this measuring principle considerably or even make it impossible.

EP 0 247 850 Al suppresses these interferences by using electrodes in which finely divided particles of the platinum metals are applied to the surface of a porous layer consisting of graphite and a porous polymeric binder. By this measure, the decomposition voltage of H2O2, which is used at > 600 mV is drastically d. H. reduced below 400 mV. Since the decomposition voltage for the redox-active interfering substances is only minimally reduced, the interference is significantly suppressed.

The platinum particles are generally applied electrochemically by redox reactions or can be accomplished by sputtering. For the manufacture of electrodes according to the principle of thick-film technology, these types of application sometimes mean a cost-intensive process modification.

Hydrogen peroxide automatically breaks down into water and oxygen. At room temperature, however, the rate of decay is immeasurably slow, but can be catalyzed by platinum group metals, for example.

As can be seen from EP 0 247 850 Al, the platinum metals catalyze the autoxidative decomposition of H2O2. wherein in the reaction (1) relevant for the H202 determination, a shift in the decomposition voltage E0 of &gt; 0.6 V on &lt; 0.4 V occurs Η2θ2 <-> 2H + + 2e '+ 02 (1)

The high conductivity of the platinum metal particles means that the catalyst and lead electrode are identical for this type of electrode

The object of the present invention is to propose, starting from the enzyme electrodes described at the outset, an amperometric enzyme electrode which is inexpensive and simple to produce and which also does not pose any problems during production using the principle of thick-film technology

This object is achieved according to the invention in that the catalytic substance of the electrode material is a stable oxide or hydrated oxide of a transition element of the fourth period of the periodic table of the elements, which is embedded in the redox-inactive conductor. Surprisingly, it was discovered that the catalysts of the oxides mentioned which are relevant for the autoxidative decay or oxide hydrates, which are poor conductors or insulators in comparison to the noble metals, also influence the oxidation of H2O2 according to equation (1) above, d. H. Lower EQ and can therefore also be used to suppress interference.

Instead of a noble metal, at least one material from the group MnO 2, FeOOH, Fe ^ O ^, Fe203, Cr203 or V2O5 can thus be used as the catalytic substance according to the invention.

Two exemplary embodiments of the invention provide that the electrode material particles of the catalytic substance, preferably Mn02. and has activated carbon particles, or that the electrode material has activated carbon particles, on the surface of which the catalytic substance, preferably MnO 2, is present in finely divided form, the particles being connected to a porous structure by a polymer binder mixed with graphite powder.

By intimately mixing z. B. manganese dioxide with graphite in an overfilled with respect to the polymer binder (ie low binder content) electrode mass, the catalyst acting as an insulator is made quasi conductive by contacting the surface with a myriad of graphite particle bridges, or by application in finely divided form on activated carbon According to the invention, the ratio of the powdery or particulate components of the electrode material to the polymer binder is at least 2: 1.

Due to the overfilling, the polymer binder only has a particle cement function, the resulting electrode material is characterized by a high porosity and thus by a large active surface.

To produce the electrode material, for. B. Mn02 powder with graphite or with activated carbon and

Graphite and with a polymer binder processed into a paste, the film of which evaporates after the -2-

AT397513B solvent has a more or less porous structure and thus has a large active surface. Furthermore, e.g. B., MnC &gt; 2 precipitated by suitable redox reactions on activated carbon and the resulting powder can be processed into a screen-printable paste analogously to the pure manganese oxide with graphite and polymer binder.

The production of an enzyme electrode according to the invention is described in more detail using manganese dioxide (manganese (IV) oxide) as an electrocatalyst. The determination of H2O2 described below in addition to the above-mentioned interfering substances was carried out at physiological pH values.

Examples (preparation of the electrode material 1) 20 parts by weight of manganese (IV) oxide powder are mixed with 100 parts by weight of graphite paste (e.g. Elektrodag 4233 SS from AchesonCoÜoiden B.V. Scheenda / Netherlands) and broken down into a homogeneous paste in a ball mill. 2) 10 parts by weight of manganese dioxide and 10 parts of activated carbon are mixed as in Example 1 with 100 parts by weight of graphite paste. 3a) Precipitation of manganese (TV) oxide on activated carbon: 10 g activated carbon are suspended in 100 ml lnNaOH and briefly heated to boiling. After cooling, the suspension is poured into a solution consisting of 1 g KMnC ^ and 500 ml ln.NaOH. Sodium sulfite dissolved in water is added with stirring until the solution has decolorized. 3b) Precipitation of MnC> 2 by synproportionation according to equation (2). 2λ4η04 · + 3Μη3 + + 40Η '- &gt; 5Mn02 + 2H20 (2) 10 g activated carbon are suspended in 200 ml ln.NaOH and briefly heated to boiling. After cooling, the suspension is introduced into a solution consisting of 0.2 g KMn04 and 200 ml water and

with a diluted ManganQOQ solution until decolourization occurs

The MnC ^ activated carbon mixtures produced according to Examples 3a) and 3b) are filtered off, washed thoroughly with water and dried at 100 ° C. 10 parts of these precipitates are mixed with 50 parts by weight of graphite paste and processed in a ball mill analogously to Example 1)

Electrode preparations

The present electrolyte materials are each applied in the form of a 1.5 x 8 mm or 2x1 mm rectangular field to a plexiglass plate printed with silver conductive tracks using a screen printing process, and the bare surfaces of the silver conductive tracks are covered with an insulating varnish

To further suppress interference of the redox-active metabolites mentioned at the outset, the electrode material of the enzyme electrode according to the invention can have a sample-side cover layer or a barrier layer which is present in the pores of the electrode material and which consists of a polyionic polymer

According to the invention it is provided that the polyionic polymer consists of polycationic polymers, polycation-forming polymers or of polymers which have both positive and negative, covalently bonded groups, for example the polycationic polymer carrying amino groups and quaternary ammonium groups, or both positive and negative Group-bearing polymer is a biopolymer, preferably a protein.

For the purpose of the interference studies, electrode surfaces were made from the paste according to Example 1). were coated, for example with polyionic polymers such as Nafion® *), albumin or polyethylenimine / -Albu-min in a ratio of 2: 1, and the albumin-containing coatings were subsequently crosslinked with glutaidialdehyde.

Electrodes that were produced in accordance with Example 3a) or 3b) were coated with 0.2 μl of a 5% glucose oxidase (GOD) solution both directly and after coating with perfluorinated Nafion® (company Aldrich-Chemie, Steinheim / FRG) and immobilized by crosslinking in glutardialdehyde vapor (30 minutes at room temperature). *) semipermeable membrane based on poly (perfluoroalkylene) sulfonic acid).

Measurements:

The individual parameters are measured potentiostatically as an counter electrode (cathode), an Ag / AgCl electrode acts.

The optimal conditions for the respective electrode were determined by varying the applied voltage. -3-

AT397 513B

Pt sheet metal, precious metal activated carbon graphite electrodes and a pure graphite electrode were measured as reference systems.

The gold-carbon electrode was produced analogously to Example 1 by mixing activated carbon, which contains 5% colloidally distributed gold, which was separated from gold chloride by a redox process. The ruthenium carbon electrode was prepared analogously, but the ruthenium was introduced into the electrode mass via ruthenium 5% on activated carbon L Platinized carbon was prepared in accordance with GB 2191 003 B (Η. P. Benetto et al.) And in the form of a 1.5 x 8 mm Electrode plate tested.

Furthermore, the electrode material was varied by replacing Mn02 with FejO ·}, Fe ^ O ^, FeOOH, and ν20 ^ as in Example 1.

H202 (1.0, 2.0 and 5.0 mM), ascorbate, p-acetamol, urate (each 1.0 mM) and glucose (2.5, 5.0, 10.0 mM), all dissolved in standard A (ISE electrolyte from AVL MEDICAL INSTRUMENTS, pH = 7.38 and physiologically relevant ionic background) are used; Standard A was also used as a reference.

The current was measured depending on the individual parameters. In Table I, Mn02 electrodes are the noble metal electrodes (Au / C, Ru / C, Pt sheet and platinized carbon) as well as a pure graphite electrode in terms of optimal voltage, change in the current flow ΔI (μΑ) with variation of the concentration of the redox-active analyte (H202 ) or the interferer, as well as the H202 concentration, which corresponds to the current flow in the presence of 1 mM interfering substance. In this measurement, the electrodes have no polyionic cover layer.

Table I ΔΙ (μΑ) when changing hourly A-analyte solution H202 concentration corresponds to 1 mM electrodes - h2o2 h2o2 Ascor- p Aceta- urate Ascor- pAce- working mat 1 mM 5 mM bat 1 mM mol 1 mM bat tamol urate span Mn02 / C according to 1 -10.0 423 - 7.0 - 2.0 -3.0 0.7 01 03 350 2 -11.0 -47.0 -15 - 1.9 -21 0.68 0.17 01 350 3a - 5.5 -25.0 - 31 - 0.9 -13 038 0.16 013 350 3b -61 -29.0 -4.0 - 1.3 -2.0 0.64 010 032 350 Au / C -0.65 - 1.65 -15.7 - 5.8 -3.4 24.2 8.90 513 500 Ru / C - 3.0 - 8.6 - 7.0 - 4.4 -2.0 233 1.46 0.66 400-500 Pt sheet -16 - 5.5 - 6.0 - 1.0 • 0.6 230 0.38 013 450-500 platinized carbon - 6.0 -32 - 3.0 0.0 -25 03 0 0.41 350 graphite -0.1 - 015 -12.0 a O o 4.0 121 100 40 600

The invention is explained in more detail below with the aid of drawings. FIG. 1 shows a schematic sectional illustration of an amperometric enzyme electrode according to the invention, FIG. 2 and FIG. 3 show details from FIG. 1, FIG. 4 shows an arrangement γοη enzyme electrodes, and FIG. 5 shows an arrangement Section along line (V * V) in Fig. 4, as well as Figs. 6a, 6b, 7a to 7c and 8 measurement diagrams with enzyme electrodes according to the invention.

1, a metallically conductive layer (2), for. B. a silver interconnect, applied on which there is an electrocatalytic, porous electrode material (4), and the above it -4-

AT 397 513 B arranged permeation-selective cover layer (5) is indicated. An insulating layer (3) prevents direct contact between the sample and the conductive layer (2).

The electrode material (4) has a porous structure and consists of activated carbon particles (10) and particles (9) of a catalytic substance, for example manganese dioxide particles (left side of FIG. 1) or of activated carbon particles (11), on the surface of which the catalytic substance finely distributed (right side of Fig. 1). The individual particles (9), (10) and (11) are cemented by a binder (8) in which graphite powder is present as the conductive pigment (8 '). The catalytic substance made of an oxide or hydrated oxide of a transition element of the fourth period is thus embedded in a redox-inactive conductor made of a binder (8) with a conductive pigment (8 '). In the pores (12) of the electrode material (4), the enzyme (13), for example glucose oxidase, is bound by adsorption or chemically fixed by crosslinking.

The cover layer (5) functioning as a permeation-selective interference barrier layer consists of a linear or cross-linked globular polymer structure (6) to which positively charged groups are covalently bound. Due to the electroneutrality relationship, these bound cations are charge compensated by means of exchangeable small negative counterions (electrostatically fixed anions (7 ')).

The chemical or electrochemical reactions taking place in the pores (12) of the porous electrode material (4) are shown schematically in FIGS. 2 and 3.

In Fig. 2 an MnÜ2 particle (9) is contacted via graphite particles (8 ') which are cemented together with a polymer binder (8). In Fig. 3, the MnC ^ particles (9) are finely distributed on the activated carbon particles (10). The enzyme, for example glucose oxidase, is identified by (13).

4 and 5 show an electrode arrangement in which on a carrier (1), for example a plexigas plate with silver conductive tracks (2), the electrode material (4) in the form of 1.5 x 8 mm or 2 x 1 mm rectangular Fields is applied. The bare surfaces of the silver conductive tracks can be covered with an insulating layer (3).

Table Π shows the influence of polyionic electrode coatings on the interference suppression on an Mn02 graphite electrode.

Table H OPTIMALE Cjj202 (111¾ corresponds to 1 mM WORKING VOLTAGE electrode ascorbate p acetamol urate (mV) MnÜ2 / C according to example 1 0.7 0.2 0.3 350 + Nafion perfluorinated (Aldrich) 0.09 0.07 0.21 340 - 350 + polyethyleneimine / albumin 2: 1-m- Glutardial-dehyd 3D cross-linked 0.21 0.08 0.07 350 + protein (bovine serum albumin cross-linked with glutaldehyde 3 Dl 0.1 0.15 0.05 350

6a and 6b show the current flow I (μΑ) versus concentration C (mM) of the redox-active parameters (o = H202, x = p. Acetate amol, + = ascorbate · = urate) measured on an Mn02 graphite electrode (a) and on an electrode prepared from platinized carbon (b).

7a to 7c the current flow I (μΑ) versus concentration C (mM) of the redox-active parameters -5-

AT397513B (same parameters as Hg. 6a, 6b) measured on an Mn02 graphite electrode (a and b) and on platinized carbon (c). The electrodes are modified as follows;

a = Mn02 / C-Nafion®-GOD

b = Mn02 / C-GOD A &gt;

c = platinized carbon Nafion -GOD

8 shows the current flow I (μ () at a voltage of 350 mV versus glucose concentration with an optimized electrode according to the invention, in which the permselective layer is applied to the electrode layer according to FIG. 1 containing the enzyme. The function of current versus glucose concentration is linear in the range from 0 to 250 mg / dl (12.5 mM).

According to the invention, the polyionized polymer can also act as a binder for the powdery or particle-shaped electrode components.

Finally, the polyionic polymer can contain the enzyme immobilized or adsorbed.

The measurements with the electrodes according to the invention have shown that MnO 2 graphite electrodes show a behavior analogous to the platinized carbon in terms of shifting the decomposition voltage, i. H. are equal in terms of electrocatalysis. It is surprising that such a strong shift cannot be observed either with platinum sheet or with ruthenium or gold activated carbon graphite electrodes.

In the case of the iron, chromium and vanadium oxide graphite electrodes not shown in Table I (production analogous to Example 1 of the Mn02 carbon electrode), a significantly weaker shift in the decomposition voltage was observed compared to Mn02, the following sequence being noted: Mn02 &gt; FeOOH &gt; Fe ^ O ^ &gt; Fe ^^ - burned &gt; Cr2Og, &gt; FejO ^ - mineral (hematite).

As can also be seen from Table I, the interference suppression also increases with the shift in the decomposition voltage.

With regard to the application or incorporation of polyionic polymers, it has been found that the anionic interferents (ascorbate, urate and the dissociated portion of p. Acetamol) z. B. by covering the electrode with a polyanionic cover layer by electrostatic repulsion to prevent the migration to the anode.

From table ersichtlich it can be seen that this effect can be achieved not only by polymers with negatively charged groups but also by predominantly positively charged polymers but also by generally polyionic polymers. In this case, the electrostatic barrier at the interface between the polymer and the sample medium seems less to play a role than the ion exchange effect of such polymers (lowering the diffusion rate in the polymer film through permanent adsorption or desorption of the interfering ions when passing through) analogous to the ion exchange chromatography. In the case of protein barrier layers, a clear interference suppression thus occurs in the interferers which are completely dissociated under physiological conditions.

Table ΙΠ as well as FIGS. 7a-7c and 8 show that, in relation to the H202 and glucose measurement in the electrode according to the invention, a partially better interference suppression is ensured in comparison with the noble metal catalysis. (Table ΙΠ follows) -6-

Claims (11)

AT 397 513 B table ΠΙ • mM glucose corresponds to 1 mM electrode ascorbate P acetamol urate MnC ^ / C + glucose oxidase immobilized + 1 + 0.7 + 0.5 Mn02 / C + Nation® + glucose oxidase immobilized -2.0 +02 0 platinized carbon + Nation® + Glucose oxidase immobilizes 0 + 5 -4 PATENT CLAIMS 1. Amperometric enzyme electrode for measuring the concentration of an enzyme substrate in a sample with an enzyme immobilized or adsorbed on or in an electrically conductive, porous electrode material, the electrode material being a redox-inactive conductor with a conductive pigment and a non-conductive binder and a finely divided catalytic substance therein, characterized in that the catalytic substance (9) of the electrode material (4) is a stable oxide or hydrated oxide of a transition element of the fourth period of the periodic table of elements, which is in the redox-inactive conductor is embedded. 2. enzyme electrode according to claim 1, characterized in that the catalytic substance (9) of the electrode material (4) from at least one material from the group Μηθ2 &gt; FeOOH, Fe ^ O ^, F ^ Oj, or V2O5. 3. Enzyme electrode according to claim 1 or 2, characterized in that the electrode material (4) particles (9) of the catalytic substance, preferably Mn02, and activated carbon particles (10), which with a graphite powder (8 ') mixed polymer binder (8) are connected to a porous structure. 4. Enzyme electrode according to claim 1 or 2, characterized in that the electrode material (4) has activated carbon particles (10), on the surface of which the catalytic substance (9), preferably Mn02, is present in finely divided form, the activated carbon particles (10) having a Graphite powder (8 ') mixed polymer binder (8) are connected to form a porous structure. 5. enzyme electrode according to one of claims 1 to 4, characterized in that the ratio of the powder or. particulate components (8 ', 9, 10) of the electrode material (4) for the polymer binder (8) is at least 2: 1 6. Enzyme electrode according to one of claims 1 to 5, characterized in that the electrode material (4) has a sample-side cover layer (5) or in the pores (12) of the electrode material (4) present barrier layer, which consists of a polyionic polymer - 7- AT 397 513 B 7. enzyme electrode according to claim 6, characterized in that the polyionic polymer consists of polycationic polymers, polycation-forming polymers or of polymers which have both positive and negative, covalently bonded groups. 8. enzyme electrode according to claim 7, characterized in that the polycationic polymer carries amino groups and quaternary ammonium groups. 9. Enzyme electrode according to claim 7, characterized in that the polymer carrying both positive and negative groups is a biopolymer, preferably a protein. 10th 10. Enzyme electrode according to one of claims 7 to 9, characterized in that the polyionic polymer acts as a binder (8) for the powdery or particulate electrode components (8 ', 9,10) 11. Enzyme electrode according to one of claims 7 to 10, characterized in that the polyionic polymer immobilizes or adsorbs the enzyme (13) contains 20 of which 3 sheets of drawings -8-